Occurrence and genetic characteristics of Giardia duodenalis in donkeys in Xinjiang, China

Giardia duodenalis is a common enteric parasite in humans and animals. To examine the occurrence and genetic characteristics of Giardia in donkeys in Xinjiang, China, 758 fecal samples from donkeys were collected, and Giardia was screened via PCR at the SSU rRNA gene. A total of 17.0% (129/758) of samples tested positive for Giardia, with the infection rate in large-scale farm and domestic donkeys being 21.4% (124/580) and 2.8% (5/178), respectively; the infection rates in <1-year-old and ≥1-year-old donkeys were 19.3% (72/374) and 12.7% (41/323), respectively. Three Giardia assemblages were identified, with assemblage B (n = 102) as the prevalent assemblage, followed by assemblage A (n = 23) and assemblage E (n = 4). Of the 129 Giardia-positive isolates, 40, 34 and 59 sequences were obtained at the bg, gdh and tpi genes, respectively. Twenty-one isolates successfully allowed multilocus genotyping (MLG), with four novel assemblage A MLGs, named MLG-AI-1 (n = 1), MLG-AI-2 (n = 1), MLG-AI-3 (n = 1), and MLG-AI-4 (n = 1) and three novel assemblage B MLGs, named MLG-B1 (n = 1), MLG-B2 (n = 14), and MLG-B3 (n = 1). Moreover, two isolates formed two MLG-mixed sequences. The results suggest that donkeys are commonly infected with Giardia in Xinjiang, and there is genetic diversity and host adaptability among the isolates.


Introduction
Giardia duodenalis is a common enteric parasite that infects various vertebrates worldwide [6].Infections can result in clinical symptoms in humans and animals, including profuse and fatty diarrhea, abdominal cramps, nausea, and wasting, representing a serious threat to human health and significant economic losses to farms [8,14].
Based on genetic sequences of Giardia at small subunit rRNA (SSU rRNA), eight assemblages (A-H) have been identified, and assemblages A and B were demonstrated to have zoonotic potential [15].In recent years, multilocus genotyping (MLG) has increasingly been used to genetically characterize Giardia isolates from humans and animals.A previous study showed that the MLG of Giardia could improve the assignment of each isolate to a specific assemblage and thereby better clarify the transmission and prevalence of giardiasis [2].
Donkeys have a long history as working animals, and they also have high livestock and medicinal value [10].Previous research conducted in partial areas of China and Iran showed that the range of Giardia infection rates was 0%-18.3%[16,20], and the prevalent assemblages were assemblages A and B. Globally, China has the largest number of donkeys (11 million), with donkey meat, skin, and milk products present ubiquitous in people's lives.To our knowledge, only two reports about Giardia detection in donkeys in China have been published, and more information on Giardia in donkeys needs to be uncovered [11,24].In this study, the aim was to understand the occurrence and genetic characteristics of Giardia in donkeys in Xinjiang, China.

Ethical standards
The protocol in this study was not required to be reviewed and approved by an Animal Studies Ethics Committee.Appropriate permission from farmers or owners was obtained before collecting fecal samples of donkeys, and no animals were harmed.

Sample collection
From July 2016 to April 2021, 758 fresh fecal samples were collected from 580 farm-raised donkeys in 13 cities and 178 free-ranging donkeys in five countryside areas of Xinjiang, China (Fig. 1).Of these, 374 samples were from young donkeys (<1 year), 323 samples were from adult donkeys (!1 year), and 61 samples were of undetermined age.All samples were collected when the donkeys defecated, in a sterile disposable latex glove and were placed in labeled sterile bags, transported and stored at 4 °C.No clinical symptoms were observed in any animal at the time of sampling.
The sampling sites involved in this study are located across Xinjiang.There are considerable differences in the environment, mode, and scale of donkey breeding between southern Xinjiang and northern Xinjiang, which are divided by the Tianshan Mountains.Southern Xinjiang is dominated by desert terrain with a dry climate and little rain, while northern Xinjiang has grassland terrain with a humid climate and abundant rain.

DNA extraction and PCR amplification
The genomic DNA of each fecal sample was extracted using a commercial E.Z.N.Stool DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA), strictly following the specifications of the manufacturer.All the extracted DNA samples were stored at À20 °C.

Sequence analysis
The positive nested PCR amplicons were sent to a commercial sequencing company (GENEWIZ, Suzhou, China) and  sequenced on an ABI PRISM™ 3730 XL DNA Analyzer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).The sequence accuracy was confirmed with bidirectional sequencing, and the sequences were aligned using ClustalX 2.1 (http://www.clustal.org/).Phylogenetic analysis was conducted using the maximum composite likelihood model, and bootstrap values were calculated by analyzing 1,000 replicates and the other chosen default parameters in MEGA 7.0 software (http://www.megasoftware.net/).

Statistical analysis
A chi-square test was performed, and 95% confidence intervals (CIs) were calculated using Crosstab in SPSS, version 24.0 (SPSS Inc., Chicago, IL, USA).Pearson's chi-squared test was used for comparisons between two groups, and p < 0.05 was considered statistically significant.

Nucleotide sequence accession numbers
The representative nucleotide sequences were submitted to GenBank at the National Center for Biotechnology Information under accession numbers tpi (OQ947877-OQ947879), gdh (OQ947880-OQ947881), and bg (OQ947882-OQ947885).

Occurrence of Giardia in donkeys
A total of 129 (17.0%)Giardia-positive fecal samples were identified by nested PCR amplification based on the SSU rRNA gene.Among the 18 sampling sites, 14 were positive for Giardia, with the highest Giardia infection rate being 36.6%(15/41) in donkeys in Pishan.The infection rates of Giardia in donkeys were statistically significant at different sampling sites (p = 0.000) (Table 2).

Sequence analysis of Giardia in donkeys
Among 129 Giardia-positive samples at the SSU rRNA gene, 23 samples were identified as Giardia assemblage A, 102 samples were identified as assemblage B, and the other 4 samples were identified as assemblage E. Assemblage B was the dominant assemblage in all Giardia-positive areas.Assemblage A was found in six areas, and assemblage E was found on only three large-scale farms (Tables 2 and 3).Assemblage B was the common assemblage identified in <1-year-old and >-year-old donkeys, and assemblage E was found only in <1-year-old donkeys (Table 3).

MLG analysis of Giardia isolates from donkeys
For the 129 Giardia-positive isolates, multilocus genotyping analysis was performed, and a total of 40, 34, and 59 sequences were obtained at the bg, gdh, and tpi genes, respectively.At the bg gene, three assemblage AI sequences and two assemblage B sequences were obtained, including two novel assemblage AI sequences and two novel assemblage B sequences.At the gdh gene, two assemblage AI sequences and three assemblage B sequences were obtained, including two novel assemblage B sequences.At the tpi gene, three assemblage AI sequences (including a novel sequence) and two assemblage B sequences (including a novel sequence) were obtained (Table 4).

Phylogenetic analysis of Giardia isolates
A phylogenetic analysis of the concatenated sequences of subassemblages A, B and A mixed with B based on three loci (bg, gdh, and tpi) was performed.Clear host separation was seen among all the samples.The vast majority of the donkey samples were isolated for the single clade, although there were two slightly different cases, one A in the same cluster as deer and the other B in the same cluster as horses (Fig. 2).Compared with the closer genetic distance to human or nonhuman primates shown by other animal hosts, including guinea pigs, sheep, deer and horses, the donkey-derived isolates in this study showed higher genetic uniqueness (Fig. 2).

Discussion
This study used the same detection methods as two other studies, but has a bigger sample size, and the Giardia infection rate was 17.0% (129/758), which was higher than the previous report in the same region (14.8%,31/210), and some other regions in China, such as Inner Mongolia (6.8%.12/176), Jilin Province (10.4%.5/48), Liaoning Province (13.8%, 4/29), and Shandong Province (14.7, 38/258) [11,24].The infection rate of Giardia in donkeys varies greatly in different regions of China, but the sample size in some regions is small [11,24]; further research is still needed.Many factors could cause differences in infection rates, such as detection methods, season, region, sampling time, and sample sizes [7].
The results of this study show that the age and feeding style of donkeys were associated with Giardia infection rates, with a higher infection rate in young animals.The Giardia infection rate in donkeys has been shown to be age-related, and the infection rate of young donkeys is higher [11,24].The same phenomenon has been seen in studies of other animals [4,6,13].In this study, the Giardia infection rate in large-scale farm donkeys was significantly higher than that in domestic donkeys.There is a lack of studies on the relationship between the prevalence of Giardia infection and feeding patterns in donkeys; previous reports have shown that the prevalence of Giardia infection is not related to feeding pattern in sheep [7].At present, studies of Giardia in donkeys are still limited around the world.In the future, it will be necessary to further strengthen the molecular epidemiological investigation of Giardia.
Giardia assemblages A, B and E were identified in this study, and assemblages A and B are two common assemblages, which is consistent with previous reports [11,24].Assemblages A and B are distributed in humans as well as several other vertebrate animals [6,14].Assemblage E was identified as the most common assemblage in ungulates [16,22] and has been found in humans [5,23].Assemblage E was identified in horses [9,20], but has rarely been reported in donkeys [11].Interestingly, assemblage E was found only in young and large-scale farm donkeys, suggesting that infection with assemblage E in donkeys may be associated with age and feeding pattern.In China, assemblages A and B of Giardia are common in humans, livestock, companion animals, wild animals, nonhuman primates, and even in wastewater, suggesting that interspecies transmission of Giardia may be common in China [7,12,17,21].The novel sequences of subassemblages A and B obtained at three genes in this study showed that Giardia in donkeys may have unique subassemblages A and B, which is in agreement with previously published conclusions [24].The MLG analysis at three genes showed the presence of four novel MLGs in assemblage AI and three novel MLGs in assemblage B. Assemblage A can be well divided into three subtypes through multiple sites, while assemblage B may have more subtypes [6].Meanwhile, previous studies have shown that mixed infections could be detected by MLG analysis [2,3], and two cases of assemblages A and B mixed infection were found in this study.Phylogenetic analysis showed that the assemblage A isolates are closer to those from deer than to those from humans, and assemblage B isolates show a higher degree of genetic similarity than other isolates, indicating that Giardia in donkeys may have unique evolutionary characteristics.

Conclusion
The results showed that Giardia is a common parasite in donkeys in Xinjiang.Young donkeys are more susceptible to Giardia, with assemblage B being the predominant assemblage.The MLG results showed that Giardia assemblages A and B from donkeys have genetic diversity and host specificity.

Figure 1 .
Figure 1.Distribution of sampling locations in southern Xinjiang, China.Filled triangles indicate sampling farms.The letters A-R represent the cities where the sampling sites were located.

2 C.
Xu et al.: Parasite 2023, 30, 50 (-)  indicates that the sequence is the same as the reference sequence.a Novel sequence.

Table 1 .
Primer sequences and reaction conditions used in nested PCR amplifications.

Table 2 .
Prevalence and distribution of assemblages of G. duodenalis infecting donkeys based on amplification and sequence analyses of the SSU rRNA gene in Xinjiang, China.

Table 3 .
Prevalence and distribution of assemblages of G. duodenalis by breeding pattern and age.

Table 4 .
Assemblage substitutions in bg, gdh, and tpi sequences within G. duodenalis assemblage A and assemblage B.

Table 5 .
Multilocus characterization of G. duodenalis isolates based on the bg, gdh, and tpi genes.